A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9–13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5′ and 3′ untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64–97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3′UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Metatranscriptomic and data mining studies have revealed a remarkable diversity of viruses infecting eukaryotic species [4,11].In addition to classical flaviviruses, a wide variety of divergent 'flavi-like' or flavivirid viruses or viral sequences have been identified outside terrestrial eukaryotes in several groups of fish [12], marine invertebrates [13] and non-bilaterians [14].Despite sharing conserved protein domain architecture, these flavi-like sequences do not form a monophyletic grouping with the four established groups of flaviviruses, instead representing a basal group to all flaviviruses.These viruses are collectively referred to as tamanaviruses in recent publications [3,14], named after the prototype species Tamana bat virus (TABV), which was isolated from Pteronotus parnellii, an insectivorous bat known as Parnell's moustached bat [15,16].
In addition to tamanavirus genomes, endogenous viral elements or 'genomic fossils' of tamanaviruses have been identified in tube-eye fish (Stylephorus) and other species [3].This suggests that tamanaviruses -like other groups within the family Flavivirus -have ancient origins in the animal kingdom.However, despite extensive studies of the viromes of vertebrates [4], with some targeting amphibians and reptiles [21,22], a significant phylogenetic gap still exists between aquatic tamanaviruses and vector-borne flaviviruses.This implies that related tamanaviruses may be found in other vertebrates, such as amphibians and reptiles, but have yet to be discovered.
We present the first discovery of a tamanavirus, Rana tamanavirus (RaTV), infecting multiple geographically distinct populations of Rana temporaria, or common frogs, in the UK.Our analysis reveals that RaTV genome organization is similar to flaviviruses but is genetically divergent and has a dinucleotide composition similar to other vertebrate tamanaviruses.Phylogenetically, RaTV defines a basal position to a putative vertebrate 'Tamanavirus' taxon.We also examined the RaTV genome, identified an Xrn1-resistant element (xrRNA/sfRNA) in the 3′ UTR, and experimentally validated this structure in vitro.

Field site selection and sample collection
During the 2014 spring breeding season (February-April) in the UK, pairs of adult R. temporaria frogs were captured during mating, and tissue samples were collected within 24 h.Before sampling, frogs were rinsed in sterile water, and a disinfectant with analgesic (Bactine; WellSpring Pharmaceutical) was applied to the distal portion of their hind limbs.Tissue samples were collected by licensed personnel who, after removing frog toe clips, placed the samples in RNAlater (Sigma Aldrich).The number of frogs sampled at each site ranged from two to 18.After sampling all animals were released.

RnA extraction, sample pooling and RnA sequencing
Toe clips from sampled R. temporaria frogs were homogenized and lysed using a Qiagen High-Frequency Tissue Lyser2 (Qiagen) with lysis buffer and stainless-steel lysis beads at 2000 Hz for 4 min.RNA was extracted from the samples using RNA isolation kits from MachereyNagel following the manufacturer's instructions.Extracted RNA was quantified using a NanoDrop and evaluated for quality using a BioAnalyser (Agilent Technologies).Only samples with RNA integrity scores >8 were chosen for pooling.Six individuals (three males and three females) from each site were pooled.The RNA concentrations of each individual within a given pool were normalized to the lowest individual, but the resulting six pools were not normalized between each other.Reverse transcription was conducted using a Superscript II kit (Invitrogen), a combination of random hexamer and oligo DT primers.Libraries were sequenced on a HiSeq2000 using v3 chemistry, generating 100 bp paired-end reads.
To recover the Poole and Deal strains of RaTV from the sequencing data, clean fastq base-called files were mapped to the Mitcham strain using Bowtie2 (v2.2.7) under default settings [31].As described previously, the depth of coverage of mapped alignment files was determined using samtools (v1.3) [32].Single-nucleotide variants of alignment files were identified using iVar (v1.2.2) [33] with a minimum quality score threshold of 20.Consensus positions were called with a minimum depth of 6.After consensus sequences were generated, pairwise comparisons between nucleotide identities were created using CLC Main Workbench (v6.9.2).A maximum-likelihood phylogeny between the three strains was generated using IQ-TREE2 using the model GTR+I+R and previously described parameters.The number of reads originating from each sample for FV3 was also determined using Bowtie2 as above and the reference accession OM927978.1.

Rana tamanavirus functional genomic annotation and E protein structural modelling
To identify the functional domains in the predicted RaTV polyprotein, we conducted a protein domain-based search using InterProScan (v5.60-92.0,available at https://www.ebi.ac.uk/interpro/search/sequence/).To predict cleavage residues of the polyprotein, we used a transmembrane topology prediction using the TMHMM Server v2.0 (https://www.cbs.dtu.dk/services/TMHMM/).To identify putative signal peptides, a 40-60 aa sliding window of the polyprotein was assessed by the SignalP v6 webserver (https://services.healthtech.dtu.dk/service.php?SignalP-6.0).To analyse dinucleotide motifs of the coding region of the RaTV genome, we added to a previous dataset of the odds ratio of dinucleotide motifs 103 Flaviviridae genomes deposited in GenBank [13].Hierarchical clustering and principal components analysis were performed using the ClustVis [34] web server (https://biit.cs.ut.ee/clustvis/).The original values were ln(x+1)-transformed before analysis.
The RaTV envelope glycoprotein monomer was modelled by ColabFold v1.5.2 [35], which utilizes AlphaFold 2 [36] using the templates in pdb70 and MMseqs2 under default conditions.The five top models were ranked based on a per-residue confidence score (pLDDT) (see Fig. S1, available in the online version of the article) and manually inspected using ChimeraX (v1.5) [37].Model number 1 was chosen for downstream visualization.The dimer was arranged using PDB:7KV8, with the domains of E coloured according to amino acid alignments and arrangement in the putative structure.

Xrn1 resistance assay
A DNA fragment corresponding to the RaTV 3′UTR under the T7 promoter was synthesized and cloned into the pIDT vector (IDT).Plasmids containing PCV 3′UTR and GFP gene fragments have been described previously [38].For in vitro transcription, plasmids were first linearized by restriction digest and purified using a Monarch PCR and DNA Clean-up Kit (NEB).3′UTRs were in vitro transcribed from 500 ng of plasmid DNA using a MEGAscript T7 Transcription Kit (Invitrogen).RNA was purified by LiCl precipitation and examined by electrophoresis in a 1 % denaturing agarose gel.RNA was then refolded in NEB3 buffer (85 °C for 5 min) and gradually cooled to 28 °C.For Xrn1 treatment, refolded RNA (1 µg) was incubated with 1 U Xrn1 (NEB) and 10 U RppH (NEB) in 20 µl of reaction mixture containing 1× NEB3 buffer (NEB) and 1 U µl -1 RNasin Plus RNase Inhibitor (Promega) for 2 h at 28 °C.The reaction was stopped by adding 20 µl of Loading Buffer II (Ambion), heating for 5 min at 85 °C and placing on ice.The denatured RNA samples were loaded into 6 % polyacrylamide TBE-Urea gels (Invitrogen), and electrophoresis was performed for 90 min in 1× TBE.The gels were stained with ethidium bromide and imaged using an OmniDoc imager (Cleaver Scientific).

RnA structure prediction
The secondary structure of the xrRNA in the 3′UTR of the RaTV gene was predicted by generating global alignments with RNAalifold consensus structure from the LocARNA package (v1.9.1) [39].The experimentally determined PCV and TABV structures provided structural constraints (#S option).Alignment scoring options were as follows: Structure Weight: 200, Indel Opening Score: −800, Indel Score: −50, Match Score: 50, Mismatch Score: 0 and alignment using RIBOSUM.The resultant secondary structures were visualized using VARNA v3.93, non-canonical C-A pairing was manually forced, and pseudoknots were located manually.

A novel tamanavirus infects the European common frog (Rana temporaria) in uK samples
During the 2014 spring breeding season (February-April) in the UK, toe clip samples were obtained from pairs of male and female adult R. temporaria frogs [40] at sites selected from the Frog Mortality Project database and a database of locations known to be free of ranaviral infections (Fig. 1a) [41][42][43][44].We conducted high-throughput transcriptome sequencing on these samples, performed de novo assembly of the resulting sequences and screened for virus-like contigs using a previously established pipeline [25].
Three of the six libraries, Mitcham, Poole and Deal, contained contigs with strong BLASTx hits to the polyprotein of TABV (Accession NP_658908.1,query cover 86 %, percentage identity 28.38 %, E-value 0, total score 955) and CLuV (Accession ATY35190.1,query cover 81 %, percentage identity 27.94 %, E-value 0, total score 896).The Mitcham library had the largest contig of 11 264nt in length.Given the closest hit to this putative virus contig, this virus was named Rana tamanavirus (RaTV).In contrast to CLuV, which encodes a single polyprotein from a predicted programmed −1 ribosomal frameshift [17], the RaTV genome contains a single ORF encoding a 3456 aa polyprotein and 5′ and 3′ UTRs of 227 and 666 nt, respectively.
We remapped the reads from all libraries to the RaTV Mitcham strain and also frog virus 3 (FV3, Ranavirus), a dsDNA virus often found in healthy frogs, and found that both disease-free and positive disease history populations contained reads that mapped to the genome of FV3 (Fig. 1b), as previously described [40].In contrast, no reads originating from RaTV were found in other libraries.The mapping coverage of the RaTV strains revealed an average coverage of 167× for Mitcham, 57× for Poole, and an

The RaTv polyprotein contains conserved Flavivirus-like protein domains and putative cleavage patterns
In the Flaviviridae, the polyprotein is multi-membrane-spanning that becomes embedded in numerous positions in endoplasmic reticulum membranes and is co-and post-translationally processed by the NS2B-NS3 chymotrypsin-like protease [45] and host proteases [46,47].The resultant conserved proteins include the structural capsid protein (C), pre-membrane protein (prM) and envelope protein (E), as well as non-structural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B and NS5.
To annotate the RaTV polyprotein, we predicted the conserved Flaviviridiae protein domains, transmembrane topology and protein cleavage motifs (Table S1) in RaTV compared to previous predictions of TABV [16] and CLuV [17] and the Flavivirus type species YFV (Fig. 2).RaTV shares conservation of protein domain architecture between putative tamanaviruses and YFV (Fig. 2).Of the structural proteins, RaTV encodes a highly conserved flavivirus E glycoprotein with central and dimerization domains (pfam00869; E-value ≤6.1E-23, position 304-548).An amino acid alignment of the RaTV glycoprotein with other tamanaviruses and YFV shows that the protein will probably resemble the domain organization of prototypical flaviviruses structurally (Fig. S2).To examine a possible structure of RaTV, the E glycoprotein was modelled using Alphafold2 Colab (Fig. 2c).Structures of E in known mature flavivirus virions consist of three ectodomains (DI-III) followed by stem and transmembrane regions anchoring E to the lipid bilayer [48][49][50].DI forms a central beta-barrel domain, which is flanked by DII, an elongated dimerization domain with the fusion peptide at the distal end.DIII is an immunoglobulin-like domain arranged on the opposite side of DI.Each of these domain features were presented in the predicted RaTV glycoprotein structure, including the fusion loop peptide, which is highly conserved in RaTV ( 382 DRGWTTGCFIFGKGGV 394 ) and is essential for viral membrane fusion [51,52].Regions of low homology between RaTV and typical vertebrate-infecting flaviviruses correlated with disordered predictions in the structure.One example of this is 267-VNVS KHDS FNNE AGSR LAGD YGYSE-291 with a disordered loop protruding from the top of DI, suggesting the structure of E in this region may be substantially different from known flaviviruses.
Generally, the host furin, putative virus NS2B-NS3-protease cleavage sites and potential host signalase sites for processing RaTV and closely related vertebrate tamanaviruses were conserved.The NS3-protease protein of flaviviruses cleaves after two basic amino acid residues (RR/RK/KR) before a small amino acid (G/A/S).
Although some predicted sites are identical to canonical NS3-protease cleavage motifs (Table S1), predicting many NS2B-NS3-protease cleavage sites for RaTV and CuLV was challenging as well-established flavivirus residues are weakly conserved in the tamanaviruses [16].Moreover, boxes 3 and 4 of the NS3-Pro domain, which have residues involved in substrate binding and recognition, showed considerable divergence.This may indicate that tamanavirus NS3-Pro may have a different substrate for cleavage that is not the canonical two basic amino acids.The Flavivirus polyproteins pre-membrane (pr) and membrane (M) proteins, processed in the trans-Golgi network, are cleaved by the host convertase furin [54], which cleaves at the highly conserved motif R-X-R/K-R.RaTV, TABV and CuLV all have perfect furin cleavage sites, so it is likely that the tamanaviruses process prM in this conserved manner.

RaTv dinucleotide composition is similar to other vertebrate tamanaviruses
Viral RNA containing CpGs are recognized by the vertebrate antiviral protein ZAP, which hinders multiplication of the virus [55].Classic CpG underrepresentation has been demonstrated in all vertebrate-infecting flaviviruses [56].In comparison, there is no clear selection against CpG dinucleotides in classical insect-only flaviviruses (cISF).
Odds ratios, which measure the expected ratio of dinucleotide composition over observed dinucleotide ratios, were calculated to determine the extent of under-or overrepresentation of dinucleotide ratios in coding sequences.An odds ratio of ≤0.78 or ≥1.23 indicates statistically significant under-or overrepresentation, respectively.The CpG motif in RaTV was found to be significantly underrepresented (0.47) at levels similar to other vertebrate (VTV, n=5) and invertebrate (ITV, n=4) tamanaviruses when compared to insect cISF CpG odds ratios (Fig. 3a).
In addition to CpG composition, the collective dinucleotide composition is reasonably predictive of viral hosts in Flaviviridae [57].To address the limitations of overinterpreting single dinucleotide motifs, the frequencies of all 16 dinucleotides were used as predictive factors for clustering analyses.Principal component analysis (PCA, Fig. 3b) and hierarchical clustering (Fig. S4) were conducted to examine the natural groupings proposed for the tamanavirus and Flavivirus groups.
The results of PCA (Fig. 3b) indicate that tamanaviruses cluster broadly together but separately from vector-borne (MBF, TBF, NKV) and cISF groups.In addition, the dendrogram of hierarchical clustering analysis (Fig. S4) demonstrates that vertebrate tamanaviruses cluster closer with vector-borne and vertebrate-infecting flaviviruses than the invertebrate tamanaviruses which were in a completely separate group independent of both VTV and MBFs.While ITV and VTV may be similar in CpG composition, the collective dinucleotide composition of both groups supports separate host-associated groups independent of the vector-borne flaviviruses and other flavivirus groups.
Using this alignment, we reconstructed a consensus maximum-likelihood phylogenetic tree using IQ-TREE2 (Fig. 4a).The resultant phylogenetic trees were robustly supported with reasonable bootstrap support values, and this tree's overall topology is congruent with previously created phylogenies [3,4].RaTV clusters basally to a clade that encompasses CuLV and TABV.We propose that tamanaviruses can be grouped into two categories based on the host species they infect.The first group, or group I tamanaviruses, includes invertebrate tamanaviruses such as embiopteran flavi-related, Waxsystermes and Wenzhou shark flavivirus [4,13,58,59].The second group, or group II tamanaviruses, includes vertebrate tamanaviruses such as TABV, CuLV and RaTV.
To investigate the genetic relationships among RaTV strains from different locations, we assembled and aligned consensus RaTV strains from Deal, Poole and Mitcham, retaining 4981 nt for analysis.Pairwise comparison of the three RaTV strains revealed a certain level of genetic diversity with 95.64-97.39% nucleotide identity (Fig. 4b).Phylogenetic analysis based on the alignment suggested that the Mitcham and Deal samples were more closely related to each other than to the RaTV strain from Poole (Fig. 4c).These findings indicate that RaTV is prevalent in UK frog samples over a geographical range of 240 km.
To determine if RaTV contains xrRNAs, we first conducted BLASTn searches and sequence scan of Rfam analysis of the 3′UTR.This analysis indicated no BLASTn similarity with any Flaviviridae 3′UTR or homology with Flavivirus Rfam families, including 3′UTR cis-acting replication element (Flavi_CRE; RF00185), Flavivirus DB element (Flavivirus_DB; RF00525) or general Flavivirus exoribonuclease-resistant RNA element (Flavi_xrRNA; RF03547).Therefore, to identify potential divergent xrRNAs, we performed a structure-and sequence-based alignment of the RaTV 3′UTR with the class 1b xrRNAs of TABV xrRNA (PDB: 7K16_P) [62,63] and the classical insect-specific flavivirus Palm Creek virus (PCV) [38] using LocARNA (Fig. 5a).This revealed a 48 nt element between 11065 and 11112, approximately 200 nt from the terminal end of the RaTV 3′UTR, which displayed homology to known class 1b xrRNA.Subsequent secondary structure modelling (Fig. 5b) indicated that this element has the typical organization of class 1b xrRNAs found in lineage 1 ISFs and TABV, with two coaxially stacked dsRNA helices P1 and P3, a pseudoknot-forming terminal loop L2, side RNA helix P2 with L1 loop, and a small internal pseudoknot.The structure also contained two non-canonical A-C pairs in the base of the P1 helix and was lacking an unpaired C between P2 and P3, which is unique for class 1b xrRNAs.However, putative RaTV xrRNA probably formed a 2 nt PK between GC-dinucleotide in L2 and the downstream sequence, which are features of class 1b xrRNAs that are also found in some pegiviruses and pestiviruses [67].This indicates that RaTV and TABV xrRNAs may have diverged from a common ancestor with cISFs, and clade-specific changes accumulated later in evolution.Given the small and unusual PK in the putative RaTV xrRNA, we then tested the structure for Xrn1 resistance.The in vitro transcribed fragment of the RaTV 3′UTR containing the putative xrRNA was treated with the recombinant Xrn1, and the digestion products were analysed by gel electrophoresis.The RNA fragments corresponding to the PCV 3′UTR containing known xrRNAs and GFP RNA that is entirely Xrn1-susceptible were treated with Xrn1 in parallel to serve as a positive and negative control, respectively.The results demonstrated that Xrn1 could not digest the 3′UTR of RaTV completely and produced a product of incomplete RNA degradation of 200 nt (Fig. 5c).The size of this product corresponds to the expected length of sfRNA based on the position of the putative xrRNA in the RaTV 3′UTR.Similar products of the incomplete RNA digestion by Xrn1 were observed with the PCV 3′UTR that corresponded in size to the three previously reported sfRNAs of this virus [38].In contrast, the GFP RNA fragment was digested without producing intermediate products.This indicates that RaTV contains a functional xrRNA in its 3′UTR and can produce sfRNA.

DISCuSSIon
In this study, we report the discovery of a novel tamanavirus that infects the European common frog.Amphibians are the most threatened group of vertebrates globally, and their populations continue to decline [68] due to a variety of threats, including habitat loss [69], climate change [70] and emerging infectious diseases (EIDs).EID threats to amphibians include fungal chytridiomycosis caused by Batrachochytrium [71] and viral pathogens from the dsDNA genus Ranavirus [72,73].Ranavirus infections cause significant adult mortality and the potential for local extinction in R. temporaria populations [74], as well as sub-lethal impacts such as altered gene expression profiles [40], changes in skin microbiome structure [75], and shifts in population genetic and demographic structure [74].
The effects of this newly discovered tamanavirus on its host and mode of transmission are currently unknown.However, RaTV infection is likely to be systemic as the virus was detected from toe clips.It is worth noting that for the related TABV, the virus has been readily recovered from the salivary glands and lungs of bats with naturally acquired infections [76].Experimental infections of closely related bat species resulted in productive infections isolated from sera, spleens, salivary glands and saliva [76], with antibodies to TABV detected by haemagglutinin inhibition and neutralization tests at 6 days.While no signs of illness were observed in any bat, experimental infections of a white-faced capuchin monkey (Cebus nigrovittatus) revealed behavioural symptoms.Infection of infant mice with TABV by the intracranial and intraperitoneal routes was also fatal.While RaTV may be a latent or asymptomatic infection in R. temporaria frogs, given the potential for coinfection of RaTV with ranaviruses and other EIDs, the complex interactions between these viruses should be examined.
In addition to showing that RaTV has divergent but structurally conserved flavivirus-like protein domains, we can demonstrate that RaTV encodes an Xrn1-resistant element in its 3′UTR.Two copies of xrRNA elements exist in the 3′UTR of TABV (GenBank Accession: MZ229974), with the tertiary structure recently solved by X-ray crystallography [63].TABV xrRNA shares conserved features with previously characterized xrRNAs and a novel set of tertiary interactions [63], informing later searches for undiscovered xrRNAs.The discovery that the same structural RNA can be achieved by very different sequences and interactions in tamanaviruses and distantly related flaviviruses has provided insight into the convergent evolution and diversity of structural RNAs within this family.While only the xrRNA elements have been characterized, additional 3′UTR elements, such as potential Flavivirus DB elements and terminal 3′ stem-loops, should be examined as additional vertebrate tamanaviruses are discovered.
In our recent study, we demonstrated that xrRNAs of this class are present in 3′UTRs of all analysed cISFs [38].Although xrRNAs of this type are common in ISFs, they are relatively rare in other flavivirus clades.For example, they do not occur in MBFs or TBFs, and within the NKV clade, they have been only found in TABV, while other NKVFs and TBFs analysed to date contained even more divergent class 2 xrRNAs.Herein, we found that RaTV contains xrRNA homologous to that of TBAV and cISFs but divergent from xrRNAs of other characterized NKVs.

Fig. 1 .
Fig. 1.Rana temporaria populations sampled for sequencing and incidence of viral infections.(a) Map of catch locations in the UK with positive history (purple) and negative history (green) of Ranavirus (FV3) infection.(B) Incidence and percentage of sequencing reads for both FV3 (blue) and the novel Rana tamanavirus (red).(C) Coverage depth of the RaTV strains from the three catch locations.Flavivirus envelope glycoprotein (E), non-structural protein 3 and 5 (NS3/5), and methyltransferase (MET) domains are indicated.

Fig. 2 .
Fig. 2. Genome architecture and E protein structure of Rana tamanavirus compared to flavivirus type species (a) yellow fever virus and (b) the putative vertebrate tamanaviruses TABV, CuLV and RaTV.Coloured boxes indicate predicted flavivirus domains: flavivirus capsid (C) and envelope glycoprotein (E), non-structural proteins 1-5 (NS1-5), peptidase (S7) and methyltransferase domain of NS5 (MET).Predicted transmembrane domains are shown with diagonal hatched lines, and predicted polyprotein cleavage residues are indicated as per the key.(c) Cartoon representation of the predicted Alphafold2 RATV E dimer.Residues corresponding to domains are coloured as follows: domain I (DI, red), domain II (DII, yellow) containing a fusion loop (green), domain III (DIII, blue), and E membrane domain containing transmembrane and perimembrane regions (orange).

Fig. 3 .
Fig. 3. Rana tamanavirus has coding sequence dinucleotide composition similar to other tamanaviruses.(a) Odds ratio calculations of the CpG motif of the coding sequence of tamanaviruses and flaviviruses.(b) Principal components analysis of the dinucleotide odds ratios of the coding sequence.Original values are ln(x+1)-transformed.Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components.Prediction ellipses bound a probability of 0.95 (n=104).Categories are as follows: cISF, classical insect-specific flavivirus; dISF, dual insect-specific flavivirus; MBF, mosquito-borne flavivirus; NKV, no known vector flavivirus; TBF, tick-borne flavivirus; MIF, marine invertebrate flavivirus; ITV, invertebrate tamanavirus; VTV, vertebrate tamanavirus.

Fig. 4 .
Fig. 4. Phylogenetic relationship of Rana tamanavirus within the Flaviviridae and Rana tamanavirus strains.(a) Maximum clade credibility tree of RaTV compared to multiple ecological Flavivirus and the informal tamanavirus taxon.RaTV is indicated with an arrowhead.Branch length represents amino acid substitutions per site with the tree rooted on the outgroup, infectious precocity virus.The GenBank accession of each protein is displayed on the label, and the tree is rooted on the infectious precocity virus, a distantly related Flaviviridae member.(b) Pairwise nucleotide identity of RaTV strains based on nucleotide differences (bottom left half) and percentage nucleotide identity (top right half).(c) Phylogenetic inference of the three assembled RaTV strains based on nucleotide sequence.Branch length represents nucleotide substitutions per site.

Fig. 5 .
Fig. 5. Genetic and biochemical characterization of the Xrn1-resistant RNA element in the RaTV 3′UTR.(a) Structure-based alignment of the RaTV 3′UTR with class 1b xrRNAs of TABV and cISF PCV analysed using LocARNA.Nucleotide conservation is shown as a bar chart, and hue shows sequence conservation of the base pair of each position.Bases are coloured based on the number of compatible base pairs C-G, G-C, A-U, U-A, G-U or U-G.Here, red colouring only shows one compatible base pair, whereas yellow and green show up to two or three compatible base pairs, respectively.The saturation of the nucleotide decreases with the number of incompatible base pairs, with the light red and yellow showing two incompatible base pairs in the alignment.(b) Predicted secondary structure of the RaTV 3′UTR xrRNA element informed from the consensus structure.Pseudoknots and non-canonical A-C base pairing are located manually.(c) An in vitro Xrn1 resistance assay with the 3′UTR of RaTV.RNA fragments corresponding to the 3′UTR of cISF PCV and GFP ORF are used as the positive and negative controls, respectively.Briefly, RNA was heated, refolded by gradual cooling to 28 °C, and then treated with purified Xrn1 and RppH (to convert 5′ triphosphate into 5′ monophosphate).The samples were then analysed by electrophoresis in a denaturing polyacrylamide gel.The gel was stained with ethidium bromide (EtBr).Ladder nucleotide sizes (L) are indicated.